VIRUS RESISTANT PLANTS

Abstract: DNA constructs encoding an RNA molecule capable of interacting with an RNA dependent RNA polymerase encoded for by a virus when invading a plant such that either an eliciting element or a plus sense RNA is produced as a consequence of the interaction with the RNA dependent RNA polymerase encoded by the said invading virus, whereby any produced plus sense RNA molecule is capable of encoding for an eliciting element, plants containing such constructs and processes for obtaining such plants.

Description: VIRUS RESISTANT PLANTS BACKGROUND The present invention relates to pathogen resistant plants and in particular to pathogen resistant plants wherein pathogen resistance is triggered in response to invading pathogens such as viruses, DNA constructs for use in such plants and methods of introducing virus induced resistance into plants. Viral infections in plants are frequently responsible for detrimental effects in growth, undesirable morphological changes, decreased yield and the like. Such infections often result in a higher susceptibility to infection in infected plants to other plant pathogens and plant pests. Virus particles generally comprise a relatively small amount of genetic material (single or double stranded RNA or DNA) protected by a protein or proteins which in some viral types can also be surrounded with host-derived lipid membranes, yielding infectious particles. Viruses are dependent on host cells for multiplication and may therefore be regarded as intracellular parasites. Plants have evolved a number of defensive mechanisms to limit the effects of viral infection. For example, so-called horizontal or partial resistances which are polygenic in nature and so-called vertical resistances which are monogenic in nature. Horizontal resistance is difficult to introduce successfully into plants in breeding programs, however, vertical resistance can be bred into plants relatively easily within plant breeding programs. Genes coding for virus resistance can act constitutively in a passive sense, ie without a requirement for inducing gene expression. Constitutively expressed virus resistances include as modes of action non-host resistances, tolerance ie inhibition of disease establishment, immunity ie inhibition of transport or the presence of antiviral agents and the like. Alternatively, genes coding for virus resistance in plants can be actively switched on by way of inducing expression of a gene or genes encoding for a viral resistance. An example of such a system includes the hypersensitive response. So-called hypersensitive responses (HSR) in plants have been reported and are generally characterized by death of plant cells in the vicinity of the penetrating pathogen shortly after infection. Movement of the pathogen through infected or invaded cells is restricted or blocked due to necrosis of the invaded cell and/or cells in the environs of the invaded cell(s). In addition, HSR involves a cascade of additional or secondary defense responses and the accumulation of certain proteins and secondary metabolites, leading to a general increased level of resistance to attack by pathogens. HSR reactions to invading organisms are generally thought to involve a resistance gene product in the plant cell which recognizes and interacts with an elicitor element, ie the product of an avirulence gene of a pathogen. Elicitor element recognition in the cells of a resistant plant triggers an HSR reaction which in its turn restricts the pathogen infection to a single cell or cells, or at most to a few plant cells in the immediate vicinity thereof. An example of HSR-mediated resistance to virus infection is that of tobacco plants harbouring the N' resistance gene to tobamoviruses such as TMV and ToMV, which contain the coat protein avirulence gene. Thus far, more than twenty single dominant HSR-type resistance genes have been identified, and are present in many agronomically important crops including tobacco, tomato, potato, pepper, lettuce, and the like. Despite the apparent abundance of resistance sources to certain viruses, many crops still lack effective resistance genes to important viral pathogens [Fraser, R.S.S. (1992). Euphytica 63:175]. Searching of wild type germplasm collections has identified only a few suitable sources of viral resistance capable of being introduced successfully into agronomically important crops. An example is the absence of vertical resistance genes to cucumber mosaic virus (CMV) in many agronomically important crop types including but not limited to tomato, pepper, cucumber, melon, lettuce and the like. Plant breeders continuously try to develop varieties of crop plant species tolerant to or resistant to specific virus strains. In the past , virus resistance conferring genes have been transferred from wild types related to commercial plants into commercial varieties through breeding. The transfer of an existing resistance in the wild from the wild type gene pool to a cultivar is a tedious process in which the resistance conferring gene(s) must first be identified in a source (donor) plant species and then combined into the gene pool of a commercial variety. Resistance or tolerance generated in this way is typically active only against one or at best a few strains of the virus in question. A further disadvantage is that the breeding programme generally takes a long time, measured in years, in getting to agronomically useful plants. In an alternative, a system referred to as "cross-protection" has been employed. Cross-protection is a phenomenon in which infection of a plant with one strain of a virus protects that plant against superinfection with a second related virus strain. The cross-protection method preferentially involves the use of avirulent virus strains to infect plants, which act to inhibit a secondary infection with a virulent strain of the same virus. However, the use of a natural cross-protection system can have several disadvantages. The method is very labour intensive because it requires inoculation of each individual plant crop, and carries the risk that an avirulent strain may mutate to a virulent strain, thus becoming a causal agent for crop disease in itself. A further possible hazard is that an avirulent virus strain in one plant species can act as a virulent strain in another plant species. Genetically engineered cross-protection is a form of virus resistance which phenotypically resembles natural cross- protection, but is achieved through the expression of genetic information of a viral coat protein from the genome of a genetically manipulated plant. It is known that expression of the tobacco mosaic virus strain UI (TMV-U1) coat protein gene from the genome of a transgenic plant can result in a delay of symptom development after infection with any TMV strain. Similarly, coat protein-mediated protection has also been obtained for alfalfa mosaic virus (AMV) , potato virus X (PVX) and cucumber mosaic virus (CMV) . For some plant viruses, eg luteoviruses, it is difficult to obtain detectable amounts of the corresponding coat protein in a transgenic plant, and consequently, virus resistance is generally lowered. Furthermore, any alleged degree of protection requires that the plant produces coat protein continually and thus imposes an energy burden on the plant. As a result of such limitations the commercial value of such technology remains unclear. A further example of genetically engineered virus resistance includes the introduction of plant viral satellite RNA wherein expression of incorporated genetic material modifies the plant virus or its effects. An object of the present invention is to provide an alternative more reliable engineered virus resistance strategy in plants to those engineered resistances known in the art, based on direct pathogen induced expression of molecules in target tissues of a plant before the invading pathogen can establish itself in the host plant. Another object of the invention is to combine genetic engineering plant transformation technology with naturally existing plant viral defense mechanisms in plant tissue. Detailed Description According to the present invention there is provided a plant virus DNA construct capable of encoding directly or indirectly for a minus sense RNA molecule capable of interacting with an RNA dependent RNA polymerase encoded for by an invading virus such that at least one eliciting element is produced as a consequence of the interaction with the RNA dependent RNA polymerase encoded by the said invading virus. In another embodiment of the invention there is provided a plant virus recombinant DNA construct capable of encoding for a plus sense RNA molecule capable of interacting with an RNA dependent RNA polymerase encoded for by an invading virus producing as a result of such interaction, a plus sense RNA molecule which is capable of encoding for at least one eliciting element capable of eliciting a natural plant defense in the plant on invasion of the plant by the said invading virus. The plant virus DNA construct can be derived from any virus source capable of attacking a plant, however it is preferred that the plant virus DNA is derived from any virus source which is known to attack, is suspected of attacking or is capable of attacking an agronomically attractive plant type. It can be a natural plant virus DNA suitably modified for expression or it may be derived synthetically. The plant virus DNA should be capable of encoding for transcription into an RNA sequence complementary (ie minus sense) to a viral RNA (ie plus sense) in plant cells. In addition, the plant virus DNA should contain a portion or segment thereof which when transcribed to yield minus sense RNA and further transcribed to plus sense RNA, upon translation of the plus sense RNA, is capable of giving rise to at least one eliciting element or part thereof sufficient to elicit a natural plant defense mechanism against an invading virus. Suitable plant virus DNA or RNA sources are those derived from plant viruses capable of invading plant types such as tomatoes, peppers, melons, lettuces, cauliflowers, broccolis, cabbages, brussels sprouts, sugar beet, corn (maize) , sweetcorn, onions, carrots, leeks, cucumbers, tobacco and the like. Also included as plant virus DNA or RNA sources are those derived from plant viruses capable of invading plant types from ornamental crops such as Impatiens, begonia, petunia, pelargoniums (geraniums, viola, cyclamen, verbena, vinca, tagetes, primula, saintpaulia and the like. A minus sense RNA molecule is one which contains at least a cistron or part thereof corresponding to at least a portion of the said plant virus DNA and is capable of giving rise to a plus sense RNA molecule transcribable from the said minus sense RNA molecule which is capable of coding for and giving rise to at least one eliciting element or part thereof in plant cells. The minus sense RNA may be directly transcribable from the said plant virus DNA or it may be transcribable from a plus sense RNA derived from the said DNA. As such, the minus sense RNA transcribable from a plus sense RNA is referred to, for the purposes of the present invention, as being indirectly transcribable from the said DNA. The orientation or polarity of the cistron or cistrons or parts thereof located on the minus sense RNA molecule can be such that the eliciting element may not be directly coded for after transcription from the plant virus DNA construct. The genetic code of the cistron or cistrons or parts thereof is located on the complementary strand to the minus sense RNA molecule ie the plus sense RNA. The cistron coding for an eliciting element becomes available for translation when the minus sense viral RNA sequence is replicated by an RNA dependent RNA polymerase encoded for by an invading virus to yield a plus sense RNA molecule. Minus sense RNA herein also includes those RNA molecules which can be described as having ambisense characteristics, such as RNA molecules from tospoviruses and the like. In such cases, the minus sense RNA contains at least a cistron corresponding to a portion of the said plant virus DNA and is capable of giving rise to a plus sense RNA transcribable from the said minus sense RNA which is capable of coding for and giving rise to at least one eliciting element or part thereof in plant cells. A plus sense viral RNA molecule is one which is capable of directly or indirectly encoding at least one eliciting element or part thereof capable of being expressed in, and having a natural or engineered plant defense eliciting activity in plant cells. A plus sense RNA molecule is also one which is complementary to a viral minus sense RNA and is capable of giving rise directly or indirectly to at least one elicitor element upon translation in plant cells. Thus, a viral sense RNA molecule can be viewed as a complementary RNA molecule to a minus sense RNA molecule. Plus sense RNA herein also includes those RNA molecules which can be described as having ambisense characteristics. In such cases, the plus sense RNA contains at least a cistron corresponding to a portion of the said plant virus DNA and is capable of directly coding for and giving rise to at least one eliciting element or part thereof in plant cells. The amount of eliciting element which is expressed in the plant cell must be sufficient to elicit at least a cellular plant defense response against an invading virus resulting in a natural or engineered plant reaction effective in blocking or restricting further viral action. Thus, plus sense RNA molecules whether they be the complement of a minus sense RNA or an ambisense RNA must be capable of giving rise to elicitor elements which are capable of triggering or eliciting a natural or engineered plant defence response, whether that be through direct translation or through interaction with a viral RNA dependent RNA polymerase (eg via a generated subgenomic RNA) . There can be one or more eliciting elements ultimately encoded by the plus sense RNA depending on the type of plant defense response/plant defence responses being elicited. The viral plus sense RNA sequence is preferably one wherein at least a viral cistron has been replaced by at least a cistron coding for an eliciting element capable of being expressed in plant cells, and having a natural or engineered plant defense eliciting activity in plant tissue. The eliciting element can be any element translatable from a plus sense RNA cistron derivable from a plant virus DNA as hereinbefore described, and can be a protein, polypeptide, or peptide or fragments thereof. Examples of preferred eliciting elements include the so-called elicitor proteins and/or cell inhibitory proteins. An elicitor protein is one which if present in plant tissue, is capable of eliciting, triggering, or inducing a hypersensitive response (HSR) , that is a natural plant defense mechanism against invading pathogens such as viruses. Elicitor proteins can be of plant virus origin, such as coat proteins, proteins involved in cell-to-cell movement, helicases, RNA- dependent RNA polymerases and the like. In addition, elicitor proteins can originate from or be derived from other plant pathogens such as bacteria, fungi, nematodes and the like. A cell inhibitory protein is a protein which if present in plant tissue, has a detrimental effect on the plant cell, leading to inhibition of cell growth eg cell division, and/or cell death. Cell inhibitory agents include but are not restricted to ribonucleases, proteinases, ribosomal inhibitory proteins, cell wall degrading proteins and the like. The minus and plus sense RNA molecules can be viewed as plant virus RNAs since they are derived from a plant DNA construct as hereinbefore described and comprise the genome or a segment of the genome of a plant virus. In such plant virus RNAs, selected nucleotide fragments can be replaced by others or can be deleted. Replacement and/or deletion of nucleotides or segments comprised of nucleotides should be such so as not to interfere with the capability of the RNA molecule to multiply or replicate in virus-infected plant cells. Also, replacement and/or deletion of nucleotides, codons or segments comprised of nucleotides should be such so as not to interfere with the ability of the RNA dependent RNA polymerase of the invading virus to recognise and act upon an RNA molecule (in plus or minus sense orientation) , and thereby initiating the sequence of events as described herein leading to the production of an effective amount of an eliciting element capable of eliciting a natural or engineered plant defense response. Examples of suitable plant virus RNA molecules include, but are not limited to genomic RNA molecules or segments thereof selected from the group comprising potyviruses, potexviruses, tobamoviruses, luteoviruses or genomic RNA or segments thereof of cucumoviruses, bromoviruses, tospoviruses and the like. The plant virus DNA is under expression control of a promoter capable of functioning in plants and includes a terminator capable of functioning in plants. A promoter is the nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Examples of promoters suitable for use in DNA constructs of the present invention include viral, fungal, bacterial, animal and plant- derived promoters capable of functioning in plant cells. A preferred promoter should express the DNA constitutively, that is in all living tissues of the plant. It will be appreciated that the promoter employed should give rise to the expression of the viral plant DNA at a rate sufficient to produce the amount of RNA capable of encoding for at least an elicitor element capable of eliciting a natural plant defense in a transformed plant on invasion of the plant by a virus. The required amount of RNA to be transcribed may vary with the type of plant. Examples of suitable promoters include the cauliflower mosaic virus 35S (CaMV 35S) and 19S (CaMV 19S) promoters, the nopaline synthase and octopine synthase promoters, the heat shock 80 (hsp80) promoter and the like. A terminator is contemplated as (A) a DNA sequence downstream of the viral DNA, coding for transcription into an RNA sequence which is capable of autocatalytical, self cleavage, to release the terminator sequences from the recombinant viral RNA sequence, followed by (B) a DNA sequence at the end of a transcriptional unit which signals termination of transcription. These elements are 3'-non-translated sequences containing polyadenylation signals, which act to cause the addition of poly adenylate sequences to the 3' end of primary transcripts. Examples of sequences mentioned under (A) include self-cleaving RNA molecules or ribozymes such as ribonuclease P, Tetrahymena L-l9 intervening sequence, hammerhead ribozymes, Hepatitis delta virus RNA, Neurospora mitochondrial VS RNA and the like [Sy ons, R.H. (1992). Ann. Rev. Biochem. 61:641]. Sequences mentioned under (B) may be isolated from funghi, bacteria, animals and/or plants. Examples, particularly suitable for use in the DNA constructs of the invention include the nopaline synthase polyadenylation signal of Agrobacterium tumefaciens, the 35S polyadenylation signal of CaMV and the zein polyadenylation signal from Zea mays . A DNA or RNA sequence is complementary to another DNA or RNA sequence if it is able to form a hydrogen-bonded complex with it, according to rules of base pairing under appropriate hybridization conditions. For the purposes of the present invention appropriate hybridization conditions may include but are not limited to, for example, an incubation for about 16 hours at 42°C, in a buffer system comprising 5 x standard saline citrate (SSC) , 0.5% sodium dodecylsulphate (SDS) , 5 x Denhardt's solution, 50% formamide and 100 μg/ml carrier DNA or RNA (hereinafter the buffer system) , followed by washing 3x in buffer comprising 1 x SSC and 0.1% SDS at 65°C for approximately an hour each time. Thus the hybridisation signal obtained for an RNA or DNA molecule, for example an autoradiogram reading, should be sufficiently clear to the man skilled in the art so as to suggest that an RNA or DNA molecule obtained could usefully be employed in the construction of plant virus DNA constructs suitable for use in the invention. Naturally, such an RNA or DNA molecule should be capable of the requisite activity as described herein. Thus replacement and/or deletion of nucleotides, codons or segments comprised of nucleotides should be such so as not to interfere with the ability of a DNA construct of the invention to code for a minus sense RNA molecule as herein described which is capable of being recognised by and of interaction with an RNA dependent RNA polymerase of an invading virus and thereby initiating the sequence of events as described herein leading to the production of an effective amount of an eliciting element capable of eliciting a natural or engineered plant defense response. Suitable hybridization conditions employed in the present invention can involve incubation in a buffer system for about 16 hours at 49°C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 55°C for about an hour each time. More preferably, hybridization conditions can involve incubation in a buffer system for about 16 hours at 55"C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 65°C for approximately an hour each time. Naturally, any RNA or DNA molecule subjected to such hybridisation conditions should be capable of the requisite activity as described herein. The invention also provides a vector capable of introducing the DNA construct of the invention into plants and methods of producing such vectors. The term vector employed herein refers to a vehicle by means of which DNA molecules or fragments thereof can be incorporated into a host organism. Suitable vehicles include plasmids, naked DNA introduced using micro- injection, particle guns, and the like [Offringa (1992) . PhD thesis, State University Leiden, The Netherlands, Chl:pages 7- 28] . The term plants as used herein is used in a wide sense and refers to differentiated plants as well as undifferentiated plant material such as protoplasts, plant cells, seeds, plantlets and the like which under appropriate conditions can develop into mature plants, the progeny thereof and parts thereof such as cuttings and fruits of such plants. The invention further provides plants comprising in their genome a DNA construct of the invention, and methods of producing such plants. The plants according to the invention have reduced susceptibility to diseases caused by the respective viruses and do not have the disadvantages and limitations of plants obtained by classical methods and genetic engineering methods as discussed herein. The invention is illustrated by the following non-limiting examples and accompanying figures. Figure 1: Schematic representation of the interaction of pathogen and plant encoded proteins leading to induction of an HSR response. Figure 2: Schematic representation of CMV resistant tobacco or tomato plants, obtained by expression of a minus-sense CMV RNA 3 molecule in which the MP gene is replaced by a gene coding for an elicitor (ToMV CP or P30) or a cell inhibitory protein (RNase TI) . Figure 3: Schematic representation of CMV resistant tobacco or tomato plants obtained by expression of a plus-sense CMV RNA 3 molecule in which the CP gene is replaced by a gene coding for an elicitor (ToMV CP or P30) or a cell inhibitory protein (RNase TI) . Sequence ID 1: Chimaeric cucumber mosaic virus RNA 3. Sequence ID 2: Coat protein of ToMV (corresponding to nucleotides from positions 123-600 of Seq. ID. No.l). Sequence ID 3: Coat protein of cucumber mosaic virus corresponding to nucleotide positions from 897-1550 of Seq. ID. No 1. Sequence ID 4: Chimaeric cucumber mosaic virus RNA 3. Sequence ID 5: RNAse TI corresponding to positions 123—437 of Seq. Id No. 4. Sequence ID 6: Chimaeric cucumber mosaic virus RNA 3, coding for P30 of ToMV. Sequence ID 7: P30 of ToMV corresponding to nucleotide positions 123-914 of Seq. ID No.7. Sequence ID 8: Chimaeric tomato spotted wilt virus S RNA, coding for the coat protein of ToMV and the non-structural protein, NSs in opposite polarity. Sequence ID 9: The non-structural protein, NSs (in opposite polarity) corresponding to nucleotide positions 1141-2543 of Seq ID No.8. Examples All CMV, TS V, and ToMV RNA-derived sequences presented here are depicted as DNA sequences for the sole purpose of uniformity. It will be appreciated that this is done for convenience only. Cultivars of Nicotiana tabacum and Lycopersicon esculentum, used in plant transformation studies, are grown under standard greenhouse conditions. Axenic explant material is grown on standard MS media [Murashige and Skoog (1962) . Physiol. Plant 15:473] containing appropriate phytohormones and sucrose concentrations. E. coli bacteria are grown on rotary shakers at 37*C in standard LB-medium. Agrobacterium tumefaciens strains are grown at 28*C in MinA medium supplemented with 0.1 % glucose [Ausubel et al., (1987). Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Inter-sciences, New York, Chichester, Brisbane, Toronto, and Singapore] . In all cloning procedures the E. coli strain >JM83 , (F " , Δ( lac-pro) , ara, rpsL, 080, dlacZM15) is used as the preferred recipient for recombinant plasmids. Binary vectors are conjugated to Agrobacterium tumefaciens strain LBA 4404, a strain containing the Ti-plasmid vir region, [Hoekema et al (1983) . Nature 303: 179] in standard triparental matings using the E. coli HB101, containing the plasmid pRK2013 as a helper strain. [Figurski and Helinski, (1979) . Proc. Natl. Acad. Sci. USA 76: 1648]. Appropriate Agrobacterium tumefaciens recipients are selected on media containing rifampicin (50 μg/ml) and kanamycine (50 μg/ml) . Cloning of fragments in the vectors pUC19 [Yanish-Perron et al (1985). Gene 33: 103], pBluescript (Stratagene) , pBIN19 [Bevan et al (1984). Nucl. Acids Res. 12: 8711] or derivatives, restriction enzyme analysis of DNA, transformation to E. coli recipient strains, isolation of plasmid DNA on small as well as large scale, nick-translation, in vitro transcription, DNA sequencing, Southern blotting and DNA gel electrophoresis are performed according to standard procedures [Maniatis et al (1982) . Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York; Ausubel et al supra, (1987)] . DNA amplification using the polymerase chain reaction (PCR) was performed as recommended by the supplier of the Taq polymerase (Perkin Elmer Cetus) . Amplification of RNA by reverse transcription and subsequent standard DNA amplification was performed using the Gene Amp RNA PCR as recommended by the supplier (Perkin Elmer Cetus) . Example 1: Isolation of CMV particles and genetic material therein A CMV serogroup I is isolated from squash and maintained on squash by mechanical passaging. Virus is purified from systemically infected squash leaves essentially following the procedure according to Francki et al [(1979) CMI/AAB Descr. of Plant Viruses 213] . Approximately 100 μg of virus in a volume of 250 μl is extracted with phenol, then with a mixture of phenol and chloroform and finally with chloroform. RNA is precipitated with ethanol and collected by centrifugation . The pellet is dissolved in 20 μl of water. Example 2: Isolation of ToMV particles and genetic material therein A ToMV isolate from tomato, is maintained on tobacco via mechanical passaging. Virus is purified from systemically infected tobacco leaves essentially following the method essentailly according to Hollings & Huttinga [ (1976) CMI/AAB Descr. of Plant Viruses 156] . Approximately 200 μg of virus in a volume of 300 μl is extracted with phenol, then with a mixture of phenol and chloroform and finally with chloroform. The RNA is precipitated with ethanol and collected by centrifugation. The pellet is dissolved in 50 μl of water. Example 3: Molecular cloning of CMV RNA 3 The sequence of RNA 3 of CMV is isolated using RNA-based PCR on purified CMV RNA (Perkin Elmer Cetus supra) . Two primers are designed, ZUP069: ( 5' TTTGGATCCA CGTGGTCTCC TTTTGGAG 3' ) , which is complementary to the first 16 nucleotides at the 3' end of RNA 3 of CMV (Seq. Id No.l), and ZUP068 : (5' TTTGGATCCG TAATCTTACC ACT 3') which is identical to the first 14 nucleotides at the 5' end of RNA 3 of CMV (Seq. Id. No.l). Both primers contain BamHl restriction sites to enable further cloning of the amplified DNA molecules. Purified CMV RNA is subjected to the Gene Amp RNA PCR, and the resulting PCR fragment is isolated from an agarose gel and cloned into Smal-linearized pUC19, yielding the recombinant plasmid pZU181. Example 4: Molecular cloning of TSWV S RNA A cDNA clone containing almost the complete TSWV S RNA-specific sequence was constructed by fusion of cDNA clones 520 and 614 on the unique EcoRl site yielding pTSWV-Sl [De Haan et al (1990) . J. Gen. Virol. 71: 1001] . The complete sequence of TSWV S RNA is isolated using RNA-based PCR on purified pTSWV-Sl DNA (Perkin Elmer Cetus supra). Two primers are designed, ZUP250: 5' (TTTGGATCCA GAGCAATCGT GTCAATTTTG TGTTCATACC TTAAC) 3' which comprises 36 nucleotides identical to the first 36 nucleotides at the 5' end of TSWV S RNA (Seq. Id. No.8), and ZUP251: 5' (TTTGGATCCA GAGCAATTGT GTCAGAATTT TGTTCATAAT CAAACCTCAC TT) 3' which comprises 43 nucleotides complimentary to the first 43 nucleotides at the 3' end of TSWV S RNA (Seq. Id. No.8). Both primers contain BamHl restriction sites to enable further cloning of the amplified DNA molecules. The resulting PCR fragment is isolated from an agarose gel and cloned into Smal-linearized pUC19, yielding the recombinant plasmid pTSWV-S2. Example 5: Molecular cloning of the CP and P30 genes of ToMV The sequence of the genes corresponding to the coat protein (CP) and P30 of ToMV is isolated using RNA-based PCR. Primer ZUP112 spans either side of the translational start codon of the CP gene of ToMV RNA: 5' GTATTAACCA TGGCTTACTC 3' (comprising 13 nucleotides identical to nucleotides 121-133 of Seq. Id. No.l) and primer ZUP113 spans either side of the translational stop codon of the CP gene of ToMV RNA: 5' GCACCCATGG ATTTAAGATG 3' (comprising 16 nucleotides complementary to nucleotides 595-610 of Seq. Id. No.l), and primer ZUP117 spans either side of the translational start codon of the P30 gene of ToMV RNA: 5 i TATTTCTCCA TGGCTCTAGT 3' (comprising 13 nucleotides identical to nucleotides 121-133 of Seq. Id No.6,) and primer ZUP118 spans either side of the translational stop codon of the P30 gene of ToMV RNA: 5' GAGTAAGCCA TGGTTAATAC 3' (comprising 13 nucleotides complementary to nucleotides 911-923 of Seq. Id. No.6) The primers contain Ncol restriction sites to enable further cloning of the amplified DNA molecules. Purified ToMV RNA is subjected to the Gene Amp RNA PCR. Resulting PCR fragments are isolated from an agarose gel and cloned into Smal-linearized pUC19, yielding the recombinant plasmids pZU183 (containing the CP gene) and pZU206 (containing the P30 gene) . Example 6: Synthesis of the ribonuclease TI gene The sequence of the gene corresponding with ribonuclease TI is synthesized on a commercial DNA synthesizer (Pharmacia LKB, Gene assembler plus) as primer ZUPllO (comprising nucleotides identical to nucleotides 121-293 of Seq. Id No. ) : 5' TTTCCATGGC ATGCGACTAC ACTTGCGGTT CTAACTGCTA CTCTTCTTCA GACGTTTCTA CTGCTCAAGC TGCCGGATAT AAACTTCACG AAGACGGTGA AACTGTTGGA TCTAATTCTT ACCCACACAA ATACAACAAC TACGAAGGTT TTGATTTCTC TGTGAGCTCT CCCTAC 3' and primer ZUPlll (comprising nucleotides complementary to nucleotides 278-446 of Seq. Id. No. ) : 5' GGGCCATGGT TATGTACATT CAACGAAGTT GTTACCAGAA GCACCAGTGT GAGTGATAAC ACCAGCATGT TGGTTGTTTT CGTTGAAGAC GACACGGTCA GCACCTGGAG AAGGACCAGA GTAAACATCA CCGCTAGAGA GGATAGGCCA TTCGTAGTAG GGAGAGCTCA C 3' Both primers contain Ncol restriction sites to enable further cloning of the amplified DNA molecules. The primers are annealed and subjected to a standard DNA PCR. The amplified DNA fragment is isolated from an agarose gel and cloned into Smal- linearized pUC19, yielding the recombinant plasmid pZU230. Example 7: Construction of an expression vector pZU-A The 35S cauliflower mosaic virus (CaMV) promoter fragment is isolated from the recombinant plasmid pZ027, a derivative of pUC19 carrying as a 444 bp Hindlll-PstI fragment the HincII- Hphl region of the 35S promoter of CaMV strain Cabb-S [Franck et al (1980) . Cell 21: 285-294] , The nucleotide sequences of CaMV strains are very similar for the different strains . The 35S promoter fragment is excised from pZ027 as a 472 bp EcoRI- Pstl fragment which contains: a part of the polylinker region, 437 bp of the non-transcribed region and the transcription initiation site and 7 bp of the non-translated leader region but not containing any 35S translational initiators. The 35S promoter fragment is ligated using T4 ligase into EcoRI-PstI linearized pZO008. The plasmid pZOOOδ carries the nopaline synthase (NOS) polyadenylation signal as a 270 bp Pstl-Hindlll fragment. The resulting recombinant plasmid pZU-A carries the 35S promoter, a unique PstI site and the NOS terminator [Gielen et al (1991) Bio/Technology 10:1363]. Example 8: Construction of a plant transformation vector, which yields a transcript which replicates upon infection with CMV. The 5' end of the minus-sense RNA 3 of CMV is fused directly to the transcription initiation site of the CaMV 35S promoter using two primers ZUP148: 5' CCACGTCTTC AAAGCAAG 3' (complementary to nucleotides of the CaMV 35S promoter), and primer ZUP146: 5'CTTCGCACCT TCGTGGGGGC TCCAAAAGGA GACCACCTCT CCAAATGAAA 3' (comprising nucleotides complementary to nucleotides 1860-1827 of Seq. Id. No.l) with pZU-A as a template in a standard DNA PCR reaction. The amplified DNA fragment is digested with EcoRV and cloned in EcoRV linearized pZU-A. The resulting plasmid is digested with BstXl and Pstl and purified on an agarose gel. pZUlδl is digested with Pstl and BstXl, the 2.1 kb insert DNA is purified on an agarose gel and subsequently cloned into the gel-purified pZU-A derivative, yielding pCMV3AS-l . The movement protein (MP) coding domain of pCMV3AS-l is replaced by a unique Ncol cloning site and the axehead structure of the Hepatitis delta viral RNA is cloned downstream of the 3' end of the minus-sense RNA 3 of CMV, by PCR amplification of two DNA fragments using pCMV3AS-l as a template. The first DNA fragment is amplified using primers ZUP050: 5' AGCTGCTAAC GTCTTATTAA G 3' (comprising nucleotides complementary to nucleotides 1020-1039 of Seq. Id. No.l) and ZUP329: 5' GTCTTTAGCA CCATGGTG 3' (comprising nucleotides identical to nucleotides 604-612 of Seq Id. No.l) The DNA fragment is digested with Nrul and Ncol and a 411 bp long DNA fragment (position 607-1016 Seq. Id. No.l) is isolated from an agarose gel. The second DNA fragment is amplified using primers ZUP327: 5' GGAGAGCCAT GGCTCGGG 3' (comprising nucleotides complementary to nucleotides 115-126 of Seq. Id. No.l) and ZUP350, a primer synthesised with nucleotides comprising nucleotides complementary to antigenomic hepatitis delta virus RNA as described by Perrotta AT & Been MD (1991) Nature Vol 350(4) pp434-436 ligated to nucleotides identical to nucleotides 1-14 (3' end of the primer) of Seq. Id. No. 1: 5'TTTCTGCAGA TCTTAGCCAT CCGAGTGGA CGTGCGTCCT CCTTCGGATG CCCAGGTCGG ACCGCGAGGA GGTGGAGATG CCATGCCGAC CCGTAATCTT ACCACT)3' . The DNA fragment is digested with Pstl and Ncol and a 208 bp. long DNA fragment is isolated from an agarose gel. Both isolated DNA fragments are cloned in pCMV3AS-l, linearized with Pstl and Nrul, to yield pCMV3AS-2. Genes coding for elicitors (example 5) or cell inhibitory proteins (example 6) can be cloned as Ncol DNA fragments into the unique Ncol site of pCMV3AS-2. The resulting pCMV3AS-2 derived plasmids are digested with Hindlll and the DNA fragments containing the chimaeric genes are isolated from an agarose gel and ligated into Hindlll linearized pBIN19, resulting in binary plant transformation vectors pBINCMV3-CP, pBINCMV3-P30 and pBINCMV3- Tl respectively. Example 9: Construction of a plant transformation vector, which yields a transcript which replicates upon infection with TSWV. The 5' end of the minus-sense TSWV S RNA is fused directly to the transcription initiation site of the CaMV 35S promoter using two primers ZUP148 (Example 8), and primer ZUP255: 5' ACACAATTGC TCTCCTCTCC AAATGAAA 3' (comprising nucleotides identical to nucleotides 2608-2621 of Seq. Id. No.8) with pZU-A as a template in a standard DNA PCR reaction. The amplified DNA fragment is digested with EcoRV and cloned in EcoR5 linearized pZU-A. The resulting plasmid is digested with Muni and Pstl and purified on an agarose gel. pTSWV-S2 is digested with Pstl and Muni, the 2.9 kb insert DNA is purified on an agarose gel and subsequently cloned into the gel-purified pZU-A derivative, yielding pTSWVSAS-1. The N coding domain of pTSWVSAS-1 is replaced by a unique Ncol cloning site and the axehead structure of the Hepatitis delta viral RNA is cloned downstream of the 3' end of the minus-sense TSWV S RNA, by PCR amplification of two DNA fragments using pTSWVSAS-1 as a template. The first DNA fragment is amplified using primers ZUP252: 5' GACCCGAAAG GGACCAATTT C 3' (comprising nucleotides complimentary to nucleotides 911-930 of Seq Id. No.8) and ZUP253: 5' TTTCCATGGC TGTAAGTTAA ATT 3' (comprising nucleotides identical to nucleotides 636-655 of Seq Id. No.8) The DNA fragment is digested with Ball and Ncol and a 269 bp long DNA fragment (position 636-911 Sequence Id No.8) is isolated from an agarose gel. The second DNA fragment is amplified using primers ZUP254: 5' TTTCCATGGT GATCGTAAAA G 3' (comprising nucleotides complementary to nucleotides 140-157 of Seq. Id No.8) and ZUP255 a primer synthesised with nucleotides comprising nucleotides complementary to antigenomic hepatitis delta virus RNA as described by Perrotta AT & Been MD (1991) Nature Vol 350(4) pp434-436, ligated to nucleotides identical to nucleotides 1-14 (3' end of the primer) of Seq. Id. No. 8: 5' TTTCTGCAGA TCTTAGCCAT CCGAGTGGAC GTGCGTCCTC CTTCGGATGC CCAGGTCGGA CCGCGAGGAG GTGGAGATGC CATGCCGACC CAGAGCAATC GTGTC 3' The DNA fragment is digested with Pstl and Ncol and a 245 bp. long DNA fragment is isolated from an agarose gel. Both isolated DNA fragments are cloned in pTSWVSAS-1, linearized with Pstl and Ball, to yield pTSWVSAS-2. Genes coding for elicitors (example 5) or cell inhibitory proteins (example 6) are cloned as Ncol DNA fragments into the unique Ncol site of pTSWVSAS-2. The resulting pTSWVSAS-2 derived plas ids are digested with Xbal and the DNA fragments containing the chimaeric genes are isolated from an agarose gel and ligated into Xbal linearized pBIN19, resulting in binary plant transformation vectors pBINTSWVS-CP (Seq Id No.8), pBINTSWVS- P30 and pBINTSWVS-Tl respectively. Example 10: Selection of suitable host plants 1) Tobacco, Nicotiana tabacum var. Samsun EN. A tobacco cultivar harboring the N' gene of N. sylvestris showing an HS response upon infection with ToMV. The CP of ToMV elicits a strong HSR defense reaction in this host. 2) Tomato, Lycopersicon esculentum var. ATV847, parental line for commercial hybrids Yaiza and Gemma. A tomato line harboring the Tπt-2 2 resistance gene to ToMV. It has been demonstrated that the P30 of ToMV elicits a HS response in this resistag30 nt genotype [Fraser (1986) CRC Crit. Rev. Plant Sci.3: 257; Keen (1990). Ann. Rev. Genet. 24: 447]. Example 11: Transformation of binary vectors to tobacco and tomato plant material Methods to transfer binary vectors to plant material are well established and known to a person skilled in the art. Variations in procedures exist due to for instance differences in used Agrobacterium strains, different sources of explant material, differences in regeneration systems depending on as well the cultivar as the plant species used. The binary plant transformation vectors as described above are used in plant transformation experiments according to the following procedures. Binary vector constructs are transferred by tri-parental mating to an acceptor Agrobacterium tumefaciens strain, followed by southern analysis of the ex-conjugants for verification of proper transfer of the construct to the acceptor strain, inoculation and cocultivation of axenic explant material with the Agrobacterium tumefaciens strain of choice, selective killing of the Agrobacterium tumefaciens strain used with appropriate antibiotics, selection of transformed cells by growing on selective media containing kanamycine, transfer of tissue to shoot-inducing media, transfer of selected shoots to root inducing media, transfer of plantlets to soil, assaying for intactness of the construct by southern analyses of isolated total DNA from the transgenic plant, assaying for proper function of the inserted chimaeric gene by northern analysis and/or enzyme assays and western blot analysis of proteins [Ausubel et al supra, (1987)]. Example 12: Expression of chimaeric sequences in tobacco and tomato plant cells RNA is extracted from leaves of regenerated plants using the following protocol. Grind 200 mg leaf material to a fine powder in liquid nitrogen. Add 800 μl RNA extraction buffer (100 mM Tris-HCl (pH 8,0), 500 mM NaCl, 2 mM EDTA, 200 mM β-Mercapto- ethanol, 0,4% SDS) and extract the homogenate with phenol, collect the nucleic acids by alcohol precipitation. Re suspend the nucleic acids in 0,5 ml 10 mM Tris-HCl (pH 8,0), 1 mM EDTA, add LiCl to a final concentration of 2 M, leave on ice for maximally 4 hours and collect the RNA by centrifugation. Re suspend in 400 μl 10 mM Tris-HCl (pH 8,0), 1 mM EDTA and precipitate with alcohol, finally re-suspend in 50 μl 10 mM Tris-HCl (pH 8,0), 1 mM EDTA. RNAs are separated on glyoxal/agarose gels and blotted to Genescreen as described by van Grinsven et al [(1986). Theor. Appl. Gen. 73:94-101]. Recombinant viral RNA sequences are detected using DNA or RNA probes labeled with [ 3 P], [ 35 S] or by using non-radioactive labeling techniques. Based on northern analysis, it is determined to what extent the regenerated plants express the chimaeric recombinant viral genes. Plants transformed with recombinant viral DNA sequences are also subjected to western blot analysis after inoculation with the respective virus. Proteins are extracted from leaves of transformed plants by grinding in sample buffer according to Laemmli [(1970). Nature 244: 29]. A 50 μg portion of protein is subjected to electrophoresis in a 12,5 % SDS-polyacrylamide gel essentially as described by Laemmli supra, (1970) . Separated proteins are transferred to nitrocellulose electrophoretically as described by Towbin et al [ (1979) . Proc. Natl. Acad. Sci. USA 76: 4350]. Transferred proteins are reacted with antiserum raised against purified ToMV particles or against purified P30 protein, according to Towbin et al supra, (1979) . Based on the results of the western analysis, it is determined that transformed plants do express elicitor proteins after inoculation with the respective virus. Example 13: Resistance of tobacco and tomato plants against CMV or TSWV infection. Transformed plants are grown in the greenhouse under standard quarantine conditions in order to prevent any infections by pathogens. The transformants are self-pollinated and the seeds harvested. Progeny plants are analyzed for segregation of the inserted gene and subsequently infected with CMV or TSWV by mechanical inoculation. Tissue from plants systemically infected with CMV or TSWV is ground in 5 volumes of ice-cold inoculation buffer (10 mM phosphate buffer) and rubbed in the presence of carborundum powder on the first two fully extended leafs of approximately 5 weeks old seedlings. Inoculated plants are monitored for symptom development during 3 weeks after inoculation. Plants containing CMV Related DNA Sequences or TSWV related DNA sequences show reduced susceptibility to CMV or TSWV infection compared with untransformed control plants which show severe systemic CMV or TSWV symptoms within 7 days after inoculation. SEQUENCE LISTING Sequence ID No.l Sequence type: Nucleotide Sequence length: 1860 nucleotides Strandness: Single stranded Molecule type: Chimaeric cucumber mosaic virus RNA 3 coding for the CP of ToMV. GTAATCTTAC CACTCGTGTG TGTGCGTGTG TGTGTGTCGA GTCGTGTTGT CCGCACATTT 60 GAGTCGTGCT GTCCGCACAT ATATTTTACC TTTTGTGTAC AGTGTGTTAG ATTTCCCGAG 120 CCATGGCTTA CTCAATCACT TCTCCATCGC AATTTGTGTT TTTGTCATCT GTATGGGCTG 180 ACCCTATAGA ATTGTTAAAC GTTTGTACAA ATTCGTTAGG TAACCAGTTT CAAACACAGC 240 AAGCAAGAAC TACTGTTCAA CAGCAGTTCA GCGAGGTGTG GAAACCTTTC CCTCAGAGCA 300 CCGTCAGATT TCCTGGCGAT GTTTATAAGG TGTACAGGTA CAATGCAGTT TTAGATCCTC 360 TAATTACTGC GTTGCTGGGG TCTTTCGATA CTAGGAATAG AATAATCGAA GTAGAAAACC 420 AGCAGAATCC GACAACAGCT GAAACGTTAG ATGCTACCCG CAGGGTAGAC GACGCTACGG 480 TTGCAATTCG GTCTGCTATA AATAATTTAG TTAATGAACT AGTAAGAGGT ACTGGACTGT 540 ACAATCAAAA TACTTTTGAA AGTATGTCTG GGTTGGTCTG GACCTCTGCA CCTGCATCTT 600 AAATCCATGG TGTATTAGTA TATAAGTATT GTGAGTCTGT ACATAATACT ATATCTATAG 660 TGTCCTGTGT GAGTTGATAC AGTAGACATC TGTGACGCGA TGCCGTGTTG AGAAGGGAAC 720 ACATCTGGTT TTAGTAAGCC TACATCACAG TTTTGAGGTT CAATTCCTCA TACTCCCTGT 780 TGAGTCCCTT ACTTTCTCAT GGATGCTTCT CCGCGAGATT GCGTTATTGT CTACTGACTA 840 TATAGAGAGT GTGTGTGCTG TGTTTTCTCT TTTGTGTCGT AGAATTGAGT CGAGTCATGG 900 A CAAATCTGA ATCAACCAGT GCTGGTCGTA ACCGTCGACG TCGTCCGCGT CGTGGTTCCC 960 GCTCCGCCCC CTCCTCCGCG GATGCTAACT TTAGAGTCTT GTCGCAGCAG CTTTCGCGAC 1020 TTAATAAGAC GTTAGCAGCT GGTCGTCCAA CTATTAACCA CCCAACCTTT GTAGGGAGTG 1080 AACGCTGTAA ACCTGGGTAC ACGTTCACAT CTATTACCCT AAAGCCACCA AAAATAGACC 1140 GTGGGTCTTA TTACGGTAAA AGGTTGTTAT TACCTGATTC AGTCACGGAA TATGATAAGA 1200 AACTTGTTTC GCGCATTCAA ATTCGAGTTA ATCCTTTGCC GAAATTCGAT TCTACCGTGT 1260 GGGTGACAGT CCGTAAAGTT CCTGCCTCCT CGGACTTATC CGTTGCCGCC ATCTCTGCTA 1320 TGTTTGCGGA CGCCGCATTT GGAGTCCAAG CTAACAACAA ATTGTTGTAT GATCTTTCGG 1380 CGGGAGCCTC ACCGGTACTG GTTTATCAGT ACATGCGCGC TGATATAGGT GACATGAGAA 1440 AGTACGCCGT CCTCGTGTAT TCAAAAGACG ATGCGCTCGA GACGGACGAG CTAGTACTTC 1500 ATGTTGACAT CGAGCACCAA CGCATTCCCA CATCTAGAGT ACTCCCAGTC TGATTCCGTG 1560 TTCCCAGAAC CCTCCCTCCG ATTTCTGTGG CGGGAGCTGA GTTGGCAGTT CTGCTATAAA 1620 CTGTCTGAAG TCACTAAACG TTTTACGGTG AACGGGTTGT CCATCCAGCT TACGGCTAAA 1680 ATGGTCAGTC GTGGAGAAAT CCACGCCAGC AGATTTACAA ATCTCTGAGG CGCCTTTGAA 1740 ACCATCTCCT AGGTTTTTTC GGAAGGACTT CGGTCCGTGT ACCTCTAGCA CAACGTGCTA 1800 GTCTTAGGGT ACGGGTGCCC CTTGTCTTCG CACCTTCGTG GGGGCTCCAA AAGGAGACCA 1860 Sequence ID No . 2 Sequence type : Amino acid Sequence length: 159 amino acids Strandness : Single stranded Molecule type : Coat protein of ToMV (corresponding to nucleotides from positions 123-599 of Seq. ID . No . l) . Met Ala Tyr Ser He Thr Ser Pro Ser Gin Phe Val Phe Leu Ser 15 Ser Val Trp Ala Asp Pro He Glu Leu Leu Asn Val Cys Thr Asn 30 Ser Leu Gly Asn Gin Phe Gin Thr Gin Gin Ala Arg Thr Thr Val 45 Gin Gin Gin Phe Ser Glu Val Trp Lys Pro Phe Pro Gin Ser Thr 60 Val Arg Phe Pro Gly Asp Val Tyr Lys Val Tyr Arg Tyr Asn Ala 75 Val Leu Asp Pro Leu He Thr Ala Leu Leu Gly Ser Phe Asp Thr 90 Arg Asn Arg He He Glu Val Glu Asn Gin Gin Asn Pro Thr Thr 105 Ala Glu Thr Leu Asp Ala Thr Arg Arg Val Asp Asp Ala Thr Val 120 Ala He Arg Ser Ala He Asn Asn Leu Val Asn Glu Leu Val Arg 135 Gly Thr Gly Leu Tyr Asn Gin Asn Thr Phe Glu Ser Met Ser Gly 150 Leu Val Trp Thr Ser Ala Pro Ala Ser 159 Sequence ID No.3 Sequence type: Amino acid Sequence length: 218 amino acids Strandness: Single stranded Molecule type: Coat protein of cucumber mosaic virus corresponding to nucleotide positions from 897-1550 of Seq. ID. No 1. Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg Arg 15 Arg Pro Arg Arg Gly Ser Arg Ser Ala Pro Ser Ser Ala Asp Ala 30 Asn Phe Arg Val Leu Ser Gin Gin Leu Ser Arg Leu Asn Lys Thr 45 Leu Ala Ala Gly Arg Pro Thr He Asn His Pro Thr Phe Val Gly 60 Ser Glu Arg Cys Lys Pro Gly Tyr Thr Phe Thr Ser He Thr Leu 75 Lys Pro Pro Lys He Asp Arg Gly Ser Tyr Tyr Gly Lys Arg Leu 90 Leu Leu Pro Asp Ser Val Thr Glu Tyr Asp Lys Lys Leu Val Ser 105 Arg He Gin He Arg Val Asn Pro Leu Pro Lys Phe Asp Ser Thr 120 Val Trp Val Thr Val Arg Lys Val Pro Ala Ser Ser Asp Leu Ser 135 Val Ala Ala He Ser Ala Met Phe Ala Asp Gly Ala Ser Pro Val 150 Leu Val Tyr Gin Tyr Ala Ala Phe Gly Val Gin Ala Asn Asn Lys 165 Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala Asp He Gly Asp Met 180 Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp Ala Leu Glu 195 Thr Asp Glu Leu Val Leu His Val Asp He Glu His Gin Arg He 210 Pro Thr Ser Arg Val Leu Pro Val 218 Sequence ID No.4 Sequence type: Nucleotide Sequence length: 1696 nucleotides Strandness: Single stranded Molecule type: Chimaeric cucumber mosaic virus RNA 3 coding for RNAse TI. GTAATCTTAC CACTCGTGTG TGTGCGTGTG TGTGTGTCGA GTCGTGTTGT CCGCACATTT 60 GAGTCGTGCT GTCCGCACAT ATATTTTACC TTTTGTGTAC AGTGTGTTAG ATTTCCCGAG 120 CCATGGCATG CGACTACACT TGCGGTTCTA ACTGCTACTC TTCTTCAGAC GTTTCTACT 180 CTCAAGCTGC CGGATATAAA CTTCACGAAG ACGGTGAAAC TGTTGGATCT AATTCTTACC 240 CACACAAATA CAACAACTAC GAAGGTTTTG ATTTCTCTGT GAGCTCTCCC TACTACGAAT 300 GGCCTATCCT CTCTAGCGGT GATGTTTACT CTGGTGGTTC TCCAGGTGCT GACCGTGTCG 360 TCTTCAACGA AAACAACCAA CTAGCTGGTG TTATCACTCA CACTGGTGCT TCTGGTAACA 420 ACTTCGTTGA ATGTACATAA CCATGGTGTA TTAGTATATA AGTATTGTGA GTCTGTACAT 480 AATACTA T CTA AGTGTC CTGTGTGAGT TGATACAGTA GACATCTGTG ACGCGATGCC 540 GTGTTGAGAA GGGAACACAT CTGGTTTTAG TAAGCCTACA TCACAGTTTT GAGGTTCAAT 600 TCCTCATACT CCCTGTTGAG TCCCTTACTT TCTCATGGAT GCTTCTCCGC GAGATTGCGT 660 TATTGTCTAC TGACTATATA GAGAGTGTGT GTGCTGTGTT TTCTCTTTTG TGTCGTAGAA 720 TTGAGTCGAG TCATGGACAA ATCTGAATCA ACCAGTGCTG GTCGTAACCG TCGACGTCGT 780 CCGCGTCGTG GTTCCCGCTC CGCCCCCTCC TCCGCGGATG CTAACTTTAG AGTCTTGTCG 840 CAGCAGCTTT CGCGACTTAA TAAGACGTTA GCAGCTGGTC GTCCAACTAT TAACCACCCA 900 ACCTTTGTAG GGAGTGAACG CTGTAAACCT GGGTACACGT TCACATCTAT TACCCTAAAG 960 CCACCAAAAA TAGACCGTGG GTCTTATTAC GGTAAAAGGT TGTTATTACC TGATTCAGTC 1020 ACGGAATATG ATAAGAAACT TGTTTCGCGC ATTCAAATTC GAGTTAATCC TTTGCCGAAA 1080 TTCGATTCTA CCGTGTGGGT GACAGTCCGT AAAGTTCCTG CCTCCTCGGA CTTATCCGTT 1140 GCCGCCATCT CTGCTATGTT TGCGGACGGA GCCTCACCGG TACTGGTTTA TCAGTACGCC 1200 GCATTTGGAG TCCAAGCTAA CAACAAATTG TTGTATGATC TTTCGGCGAT GCGCGCTGAT 1260 ATAGGTGACA TGAGAAAGTA CGCCGTCCTC GTGTATTCAA AAGACGATGC GCTCGAGACG 1320 GACGAGCTAG TACTTCATGT TGACATCGAG CACCAACGCA TTCCCACATC TAGAGTACTC 1380 CCAGTCTGAT TCCGTGTTCC CAGAACCCTC CCTCCGATTT CTGTGGCGGG AGCTGAGTTG 1440 GCAGTTCTGC TATAAACTGT CTGAAGTCAC TAAACGTTTT ACGGTGAACG GGTTGTCCAT 1500 CCAGCTTACG GCTAAAATGG TCAGTCGTGG AGAAATCCAC GCCAGCAGAT TTACAAATCT 1560 CTGAGGCGCC TTTGAAACCA TCTCCTAGGT TTTTTCGGAA GGACTTCGGT CCGTGTACCT 1620 CTAGCACAAC GTGCTAGTCT TAGGGTACGG GTGCCCCTTG TCTTCGCACC TTCGTGGGGG 1680 CTCCAAAAGG AGACCA 1696 Sequence ID No. 5 Sequence type: Amino Acid Sequence length: 105 amino acids Strandness: Single stranded Molecule type: RNAse TI corresponding to positions 123-437 of Seq. Id No. 4. Met Ala Cys Asp Tyr Thr Cys Gly Ser Asn Cys Tyr Ser Ser Ser 15 Asp Val Ser Thr Ala Gin Ala Ala Gly Tyr Lys Leu His Glu Asp 30 Gly Glu Thr Val Gly Ser Asn Ser Tyr Pro His Lys Tyr Asn Asn 45 Tyr Glu Gly Phe Asp Phe Ser Val Ser Ser Pro Tyr Tyr Glu Trp 60 Pro He Leu Ser Ser Gly Asp Val Tyr Ser Gly Gly Ser Pro Gly 75 Ala Asp Arg Val Val Phe Asn Glu Asn Asn Gin Leu Ala Gly Val 90 He Thr His Thr Gly Ala Ser Gly Asn Asn Phe Val Glu Cys Thr 105 Sequence ID No.6 Sequence type: Nucleotide Sequence length: 2173 nucleotides Strandness: Single stranded Molecule type: Chimaeric cucumber mosaic virus RNA 3, coding for P30 of ToMV. GTAATCTTAC CACTCGTGTG TGTGCGTGTG TGTGTGTCGA GTCGTGTTGT CCGCACATTT 60 GAGTCGTGCT GTCCGCACAT ATATTTTACC TTTTGTGTAC AGTGTGTTAG ATTTCCCGAG 120 CCATGGCTCT AGTTGTTAAA GGTAAGGTAA ATATTAATGA GTTTATCGAT CTGTCAAAGT 180 CTGAGAAACT TCTCCCGTCG ATGTTCACGC CTGTAAAGAG TGTTATGGTT TCAAAGGTTG 240 ATAAGATTAT GGTCCATGAA AATGAATCAT TGTCTGAAGT AAATCTCTTA AAAGGTGTAA 300 AACTTATAGA AGGTGGGTAT GTTTGCTTAG TTGGTCTTGT TGTGTCCGGT GAGTGGAATT 360 TCCCAGATAA TCGCCGTGGT GGTGTGAGTG TCTGCATGGT TGACAAGAGA ATGGAAAGAG 420 CGGACGAAGC CACACTGGGG TCATATTACA CTGCTGCTGC TAAAAAGCGG TTTCAGTTTA 480 AAGTGGTCCC AAATTACGGT ATTACAACAA AGGATGCAGA AAAGAACATA TGGCAGGTCT 540 TAGTAAATAT TAAAAATGTA AAAATGAGTG CGGGCTACTG CCCTTTGTCA TTAGAATTTG 600 TGTCTGTGTG TATTGTTTAT AAAAATAATA TAAAATTGGG TTTGAGGGAG AAAGTAACGA 660 GTGTGAACGA TGGAGGACCC ATGGAACTTT CGGAAGAAGT TGTTGATGAG TTCATGGAGA 720 ATGTTCCAAT GTCGGTTAGA CTCGCAAAGT TTCGAACCAA ATCCTCAAAA AGAGGTCCGA 780 AAAATAATAA TAATTTAGGT AAGGGGCGTT CAGGCGGAAG GCCTAAACCA AAAAGTTTTG 840 ATGAAGTTGA AAAAGAGTTT GATAATTTGA TTGAAGATGA AGCCGAGACG TCGGTCGCGG 900 ATTCTGATTC GTATTAACCA TGGTGTATTA GTATATAAGT ATTGTGAGTC TGTACATAAT 960 ACTATATCTA TAGTGTCCTG TGTGAGTTGA TACAGTAGAC ATCTGTGACG CGATGCCGTG 1020 TTGAGAAGGG AACACATCTG GTTTTAGTAA GCCTACATCA CAGTTTTGAG GTTCAATTCC 1080 TCATACTCCC TGTTGAGTCC CTTACTTTCT CATGGATGCT TCTCCGCGAG ATTGCGTTAT 1140 TGTCTACTGA CTATATAGAG AGTGTGTGTG CTGTGTTTTC TCTTTTGTGT CGTAGAATTG 1200 AGTCGAGTCA TGGACAAATC TGAATCAACC AGTGCTGGTC GTAACCGTCG ACGTCGTCCG 1260 CGTCGTGGTT CCCGCTCCGC CCCCTCCTCC GCGGATGCTA ACTTTAGAGT CTTGTCGCAG 1320 CAGCTTTCGC GACTTAATAA GACGTTAGCA GCTGGTCGTC CAACTATTAA CCACCCAACC 1380 TTTGTAGGGA GTGAACGCTG TAAACCTGGG TACACGTTCA CATCTATTAC CCTAAAGCCA 1440 CCAAAAATAG ACCGTGGGTC TTATTACGGT AAAAGGTTGT TATTACCTGA TTCAGTCACG 1500 GAATATGATA AGAAACTTGT TTCGCGCATT CAAATTCGAG TTAATCCTTT GCCGAAATTC 1560 GATTCTACCG TGTGGGTGAC AGTCCGTAAA GTTCCTGCCT CCTCGGACTT ATCCGTTGCC 1620 GCCATCTCTG CTATGTTTGC GGACGCCGCA TTTGGAGTCC AAGCTAACAA CAAATTGTTG 1680 TATGATCTTT CGGCGGGAGC CTCACCGGTA CTGGTTTATC AGTACATGCG CGCTGATATA 1740 GGTGACATGA GAAAGTACGC CGTCCTCGTG TATTCAAAAG ACGATGCGCT CGAGACGGAC 1800 GAGCTAGT C TTCATGTTGA CATCGAGCAC CAACGCATTC CCACATCTAG AGTACTCCCA 1860 GTCTGATTCC GTGTTCCCAG AACCCTCCCT CCGATTTCTG TGGCGGGAGC TGAGTTGGCA 1920 GTTCTGCTAT AAACTGTCTG AAGTCACTAA ACGTTTTACG GTGAACGGGT TGTCCATCCA 1980 GCTTACGGCT AAAATGGTCA GTCGTGGAGA AATCCACGCC AGCAGATTTA CAAATCTCTG 2040 AGGCGCCTTT GAAACCATCT CCTAGGTTTT TTCGGAAGGA CTTCGGTCCG TGTACCTCTA 2100 GCACAACGTG CTAGTCTTAG GGTACGGGTG CCCCTTGTCT TCGCACCTTC GTGGGGGCTC 2160 CAAAAGGAGA CCA 2173 Sequence ID No.7 Sequence type: Amino acid Sequence length: 264 amino acids Strandness: Single stranded Molecule type: P30 of ToMV corresponding to nucleotide positions 123-914 of Seq. ID No.6. Met Ala Leu Val Val Lys Gly Lys Val Asn He Asn Glu Phe He 15 Asp Leu Ser Lys Ser Glu Lys Leu Leu Pro Ser Met Phe Thr Pro 30 Val Lys Ser Val Met Val Ser Lys Val Asp Lys He Met Val His 45 Glu Asn Glu Ser Leu Ser Glu Val Asn Leu Leu Lys Gly Val Lys 60 Leu He Glu Gly Gly Tyr Val Cys Leu Val Gly Leu Val Val Ser 75 Gly Glu Trp Asn Phe Pro Asp Asn Arg Arg Gly Gly Val Ser Val 90 Cys Met Val Asp Lys Arg Met Glu Arg Ala Asp Glu Ala Thr Leu 105 Gly Ser Tyr Tyr Thr Ala Ala Ala Lys Lys Arg Phe Gin Phe Lys 120 Val Val Pro Asn Tyr Gly He Thr Thr Lys Asp Ala Glu Lys Asn 135 He Trp Gin Val Leu Val Asn He Lys Asn Val Lys Met Ser Ala 150 Gly Tyr Cys Pro Leu Ser Leu Glu Phe Val Ser Val Cys He Val 165 Tyr Lys Asn Asn He Lys Leu Gly Leu Arg Glu Lys Val Thr Ser 180 Val Asn Asp Gly Gly Pro Met Glu Leu Ser Glu Glu Val Val Asp 195 Glu Phe Met Glu Asn Val Pro Met Ser Val Arg Leu Ala Lys Phe 210 Arg Thr Lys Ser Ser Lys Arg Gly Pro Lys Asn Asn Asn Asn Leu 225 Gly Lys Gly Arg Ser Gly Gly Arg Pro Lys Pro Lys Ser Phe Asp 240 Glu Val Glu Lys Glu Phe Asp Asn Leu He Glu Asp Glu Ala Glu 255 Thr Ser Val Ala Asp Ser Asp Ser Tyr 264 Sequence ID No.8 Sequence type: Nucleotide Sequence length: 2621 nucleotides Strandness: Single stranded Molecule type: Chimaeric tomato spotted wilt virus S RNA, coding for the coat protein of ToMV and the non-structural protein, NSs in opposite polarity. AGAGCAATCG TGTCAATTTT GTGTTCATAC CTTAACACTC AGTCTTACAA ATCATCACAT 60 TAAGAACCTA AGAAACGACT GCGGGATACA GAGTTGCACT TTCGCACCTT GAGTTACATA 120 CGGTCAAAGC ATATAACAAC TTTTACGATC ACCATGGCTT ACTCAATCAC TTCTCCATCG 180 CAATTTGTGT TTTTGTCATC TGTATGGGCT GACCCTATAG AATTGTTAAA CGTTTGTACA 240 AATTCGTTAG GTAACCAGTT TCAAACACAG CAAGCAAGAA CTACTGTTCA ACAGCAGTTC 300 AGCGAGGTGT GGAAACCTTT CCCTCAGAGC ACCGTCAGAT TTCCTGGCGA TGTTTATAAG 360 GTGTACAGGT ACAATGCAGT TTTAGATCCT CTAATTACTG CGTTGCTGGG GTCTTTCGAT 420 ACTAGGAATA GAATAATCGA AGTAGAAAAC CAGCAGAATC CGACAACAGC TGAAACGTTA 480 GATGCTACCC GCAGGGTAGA CGACGCTACG GTTGCAATTC GGTCTGCTAT AAATAATTTA 540 GTTAATGAAC TAGTAAGAGG TACTGGACTG TACAATCAAA ATACTTTTGA AAGTATGTCT 600 GGGTTGGTCT GGACCTCTGC ACCTGCATCT TAAATCCATG GCTGTAAGTT AAATTATAAA 660 AAAGCCTATA AATATATAAA GCTTTCTTTA TCTTTATTGC TTGTGCTTGC TTAGTGTGTT 720 AAATTTTAAA TAAGTGTGTT TAATTAAAGT TTGCTTTCTG TGTGTTGTGC TTAATAAATA 780 ATAAAATAAC AAAAACAACG AAAACAAAAA ATAAATAAAA TAAAAATAAA ATAAAAATAA 840 AATAAAATAA AAATAAAATA AAAAATAAAA AACAAAAAAC AAAAAACAAA AACAAAACCC 900 AAATTTGGCC AAATTGGTCC CTTTCGGGTC TTTTTGGTTT TTCGTTTTTT AATTTTTTGT 960 TGTTTTTATT TCATTTTTTG ATTTTATTTT ATTTTAATTT TATTTTCATT TTTATTTTTT 1020 GTTTTTATGG TTTCTACTAG ACAGGAGGAA TTTGAAAGAG ATGACAAACA GAGAAATAAT 1080 TATAAGTAAA GAAAGAAAAT AAACATAACA TAATTAGAAA AAGCTGGACA AAGCAAGATT 1140 ATTTTGATCC TGAAGCATAC GCTTCCTTAA CCTTAGATTC TTTCTTTTTG ATCCCGCTTA 1200 AATCAAGCTT TAACAAAGAT TTTGCAACTG AAATAGATTG TGGAGAAATT TTAATTTCTC 1260 CTCTGGCAAA GTCTATCTTC CATGAAGGGA TTTGGATGCT GTCTAAGTAA GACATAGTTT 1320 GTGTGTTAGA TGGAAGACAT TCAAGTGTTT TTGAAAGGAA ATATTTCCTT TTGTAGGCAT 1380 CTTCACTGTA ATTCAAGGTT CTTTCACCTA AATCTAACTT. TCCAGGAGTT AGCTCAAGGT 1440 TGTTCAAAGT GTAGATGATT ACATCTTCTT GCAAGTTAGT TGCAAAGAAC TTGTGCAAAG 1500 ATGTGTGAGT TTCGAGCCAG AGCATTGGAA CCGATCCTTT GGGGTATGAA GGGTCATGAA 1560 CAATGTTGTA AGGCTCCTTT AAATCAGAAA ACATCATTGA TAATTCAAAA GGAGCTTTGC 1620 ATTTGCGAAT TGGGAGCTGA TGCTTGCAAA TAACAGTAAT GTTTAAAGCT GTCTCAACAC 1680 TGTTATGGTT TGGAATGCAG GCAATAGATA AATAAAATGT TTTGTTTGTT TCATCTCCTG 1740 CAACCTTGAA CAATTTCTGA ATGGAAACCT GCTTCAAAAC CTTTGGAACC CTTAGCCAGA 1800 GGCTCAGCTT GAAATGAGAA TCAGTGGAAG CTTGAGAGTT AGGCATGATG TTGTTTTCTG 1860 CTGACATGAG CAGAGATTTC ACTGCAAGAG AATTTACAGT TCTGTTGTTG CTTTCAACTT 1920 GATTGAAATT TGGCTTGAAA CTGTACAGCC ATTCATGGAC ATTTCTGTTA GGAGATAGAA 1980 CATTCACTTT GCCTAAAGCC TGATTATAGC ACATCTCGAT CTTATAGGTA TGCTCTTTGA 2040 CACAAGACAA AGAGCCTTTG TTTGCAGCTT CAATGTATTT GTCATTGGGA ATTATGTCTT 2100 TTTCTTGGAG CTGGAATCGG TCTGTAATAT CAGATCTGTT CATGATAGAT TCAATAGAGT 2160 GGAGCTGGGC AGGAGACAAA ACCTTCAAAT GACCTTGATG TTTCACTCCG TTAGCATTGA 2220 CTGTATTTGA GCAAACAGAT AGTGCCAGAA CAGAGTTATC AATATTGATG CTAAAATCAA 2280 TATCATCAAA AATAGGGATA TACACATGCT GAGAAAGAAA TCTCTTCTTC TTCACAGGGA 2340 AGATCCCTAC TTTGCAGTAT AGCCAAAGGA CTACTTTGCT TCTTGAATCA GAATACAGCT 2400 GGGTCTGAAC TAGTTGAGAA CCAGTACCAA GTTCATGAAT CCAGTAAGAA TCTACAACAG 2460 CTTTACCAGA TGCAGTTGAT CCCCAGACTG AAGCTCTTGT CTGAATGATC GACTCATAAA 2520 CACTTGAAGA CATTATGGTT ATTGGTACTG TGTTCTTATT ACAGTATTGT GATTTTCTAA 2580 GTGAGGTTTG ATTATGAACA AAATTCTGAC ACAATTGCTC T 2621 Sequence ID No.9 Sequence type: Amino acid Sequence length: 464 amino acids Strandness: Single stranded Molecule type: The non-structural Protein, NSs corresponding to nucleotide positions 1142-2533 (in opposite polarity) of Seq ID No.8. Met Ser Ser Ser Val Tyr Glu Ser He He Gin Thr Arg Ala Ser 15 Val Trp Gly Ser Thr Ala Ser Gly Lys Ala Val Val Asp Ser Tyr 30 Trp He His Glu Leu Gly Thr Gly Ser Gin Leu Val Gin Thr Gin 45 Leu Tyr Ser Asp Ser Arg Ser Lys Val Val Leu Trp Leu Tyr Cys 60 Lys Val Gly He Phe Pro Val Lys Lys Lys Arg Phe Leu Ser Gin 75 His Val Tyr He Pro He Phe Asp Asp He Asp Phe Ser He Asn 90 He Asp Asn Ser Val Leu Ala Leu Ser Val Cys Ser Asn Thr Val 105 Asn Ala Asn Gly Val Lys His Gin Gly His Leu Lys Val Leu Ser 120 Pro Ala Gin Leu His Ser He Glu Ser He Met Asn Arg Ser Asp 135 He Thr Asp Arg Phe Gin Leu Gin Glu Lys Asp He He Pro Asn 150 Asp Lys Tyr He Glu Ala Ala Asn Lys Gly Ser Leu Ser Cys Val 165 Lys Glu His Thr Tyr Lys He Glu Met Cys Tyr Asn Gin Ala Leu 180 Gly Lys Val Asn Val Leu Ser Pro Asn Arg Asn Val His Glu Trp 195 Leu Tyr Ser Phe Lys Pro Asn Phe Asn Gin Val Glu Ser Asn Asn 210 Arg Thr Val Asn Ser Leu Ala Val Lys Ser Leu Leu Met Ser Ala 225 Glu Asn Asn He Met Pro Asn Ser Gin Ala Ser Thr Asp Ser His 240 Phe Lys Leu Ser Leu Trp Leu Arg Val Pro Lys Val Leu Lys Gin 255 Val Ser He Gin Lys Leu Phe Lys Val Ala Gly Asp Glu Thr Asn 270 Lys Thr Phe Tyr Leu Ser He Ala Cys He Pro Asn His Asn Ser 285 Val Glu Thr Ala Leu Asn He Thr Val He Cys Lys His Gin Leu 300 Pro He Arg Lys Cys Lys Ala Pro Phe Glu Leu Ser Met Met Phe 315 Ser Asp Leu Lys Glu Pro Tyr Asn He Val His Asp Pro Ser Tyr 330 Pro Lys Gly Ser Val Pro Met Leu Trp Leu Glu Thr His Thr Ser 345 Leu His Lys Phe Phe Ala Thr Asn Leu Gin Glu Asp Val He He 360 Tyr Thr Leu Asn Asn Leu Glu Leu Thr Pro Gly Lys Leu Asp Leu 375 Gly Glu Arg Thr Leu Asn Tyr Ser Glu Asp Ala Tyr Lys Arg Lys 390 Tyr Phe Leu Ser Lys Thr Leu Glu Cys Leu Pro Ser Asn Thr Gin 405 Thr Met Ser Tyr Leu Asp Ser He Gin He Pro Ser Trp Lys He 420 Asp Phe Ala Arg Gly Glu He Lys He Ser Pro Gin Ser He Ser 435 Val Ala Lys Ser Leu Leu Lys Leu Asp Leu Ser Gly He Lys Lys 450 Lys Glu Ser Lys Val Lys Glu Ala Tyr Ala Ser Gly Ser Lys 464

Claims: CLAIMS 1. A DNA construct capable of encoding directly or indirectly for a minus sense RNA molecule capable of interacting with an RNA dependent RNA polymerase encoded for by a virus when invading a plant such that at least one eliciting element is produced as a consequence of the interaction with the RNA dependent RNA polymerase encoded by the said invading virus, which construct is under expression control of a promoter and a terminator capable of functioning in plants. 2. A recombinant DNA construct capable of encoding for a plus sense RNA molecule capable of interacting with an RNA dependent RNA polymerase encoded for by a virus when invading a plant and producing as a result of such interaction a plus sense RNA molecule which is capable of encoding for at least one eliciting element. 3. A construct according to claim 1 comprising Seq. Id. No. 1 4. A construct according to claim 1 comprising Seq. Id. No. . 5. A construct according to claim 1 comprising Seq. Id. No.6. 6. A construct according to claim 1 comprising Seq. Id. No.8. 7. A recombinant DNA construct according to claim 1 or claim 2 wherein the eliciting element is selected from the group comprising proteins, polypeptides or peptides. 8. A construct according to Claim 7 wherein the eliciting element is capable of eliciting a hypersensitive response or the release of a cell inhibitory protein in a plant. 9. A construct according to Claim 1 or Claim 2 wherein the elicitor protein is of plant virus, bacterial, fungal or nematode origin. 10. A construct according to Claim 1 or Claim 2 wherein the cell inhibitory protein is selected from the group comprising ribonucleases, proteinases, ribosomal inhibitory proteins, and cell wall degrading proteins. 11. A construct according to any one of Claims 1 to 10 wherein the construct comprises a constitutive promoter selected from the group consisting of viral, fungal, bacterial and plant derived promoters. 12. A construct according to Claim 11 wherein the promoter is selected from the group consisting of CaMV 19S, nopaline synthase, octopine synthase, heat shock 80 promoters. 13. Plants containing in their genome a construct according to any one of Claims 1 to 12. 14. A process of preparing plants according to Claim 13, which comprises: A) Inserting into the genome of a plant cell a DNA construct according to Claim 1; B) obtaining transformed cells; C) regenerating from the transformed cells genetically transformed plants.

Inventors: DE HAAN PETRUS THEODORUS, DE HAAN, PETRUS, THEODORUS
Applicants: HAAN PETRUS THEODORUS DE, SANDOZ AG, SANDOZ LTD, DE HAAN, PETRUS, THEODORUS, SANDOZ LTD., SANDOZ-ERFINDUNGEN VERWALTUNGSGESELLSCHAFT M.B.H., SANDOZ-PATENT-GMBH
Application year: 1994
Main classification: C12N15
IPC codes: A01H1, C12N15